Recording the electrical impulses of many individual neurons in intact brain circuits in real time would enable a detailed understanding of how the brain processes information. Technologies for high-fidelity large-scale voltage sensing with cellular resolution would also provide new high-resolution methods for analyzing for how diseases of the brain impact circuit function. However, current methods lack the ability to detect the rich variety of electrical impulses in large numbers of neurons in deep locations in the brain. Imaging of activity-induced calcium transients using genetically encoded calcium indicators and two-photon (2P) microscopy have led to tremendous progress in our understanding of how individual neurons and neuronal populations participate in information representation and circuit plasticity. This now-established paradigm has demonstrated the benefits of combining genetically encoded optical reporters, which allow for investigation of specific neuronal types over time and with minimal perturbation, with 2P microscopy, which enables recording of optical signals in multiple neurons through hundreds of microns of brain tissue. We aim to develop a new paradigm for activity imaging using genetically encoded voltage indicators (GEVIs) and fast 2P imaging. In contrast to calcium indicators, voltage indicators can provide information on subthreshold voltage changes, which form the basis of neuronal computation and modulate excitability, and on timing and order of neuronal action potential firing events, all basic essential information required for understanding information processing in brain circuits. However, in vivo voltage imaging is currently limited by two constraints. First, further improvements to GEVI brightness, responsivity, wavelengths, and localization would ease the detection of electrical events. Second, the speed of conventional 2P imaging is insufficient for large-scale voltage imaging with single-cell resolution. In this project, we will optimize the recording of electrical activity from many individual neurons in the brain at high speed and at depth, integrating the expertise of four groups with expertise in GEVI engineering, fast 2P scanning methods, and systems neuroscience. Our approach combines optimization of the GEVI class that currently responds best to 2P illumination with improvement of fast random-access multi-photon scanning methods that can capture these fast optical signals from large numbers of neurons. Specifically, we will carry out the following aims: (1) Development of ASAP-family GEVIs along axes of brightness, responsivity, wavelength, and subcellular localization, and comprehensive validation in brain tissue under 2P illumination, (2) development of third-generation (3G) RAMP with motion correction and enhanced throughput via light patterning and excitation and emission multiplexing, and (3) recording voltage from hundreds of neurons in vivo with 1ms precision with RAMP microscopy and GEVIs.